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Abstract

Establishment and maintenacne of a polarized axis is essential for all organisms. Cells can either change their shape in response to extracellular cues or maintain a stable polarity axis via landmarks defined in relation to internal cues. In the fission yeast Schizosaccharomyces pombe,microtubules
regulate
cortical cell
polarity
together with
the landmark
protein Tea1.
Tea1 is
transported
to
cell
tips
on
microtubule
plus-­‐ends
and
deposited
upon
microtubule
contact
with
the
membrane.
Although
Tea1
has
been
shown
to
interact
with
several
binding-­partners, Tea1
anchoring at the
cell tip
depends
mostly
on
the
membrane-­associated
protein,
Mod5.
Tea1
and
Mod5
accumulate
in
clusters
at
the
cell
tip
in
a
mutually
dependent
manner. I
used
a
combination
of
live-­‐cell
imaging,
FRAP
(Fluorescence
Recovery
After
Photobleaching)
and
computational
modeling
to
dissect
the
dynamics
of
the
Tea1-­‐Mod5
interaction.
I
have
shown
that
although
Tea1
is
stably
associated
with
the
cell
tip,
Mod5
is
mobile
within
the
cell
tip.
I
proposed
a
model
in
which
Tea1
is
stable
at
the
cell
tip
due
to
self-­‐polymerization
and
association
in
the
form
of
a
cluster-­‐network.
In
the
model,
the
role
of
Mod5
in
the
cluster-­‐network
is
to
facilitate
the
formation
of
Tea1-­‐Tea1
interactions.
Moreover,
in
the
model,
Mod5
is
restricted
to
the
cell
tip
due
to
iterative
binding
to
and
release
from
the
Tea1
cluster-­‐network.
The
properties
of
the
proposed
Tea1
cluster-­‐
network
might
contribute
to
the
behavior
of
Tea1
as
a
polarity
landmark. I
hypothesized
that
Tea1
transfer
from
the
microtubules
to
the
cell
tip
was
regulated
by
phosphorylation.
Tea1
phosphorylated
residues
were
mapped
using
mass
spectroscopy
(MS),
and
identified
to
be
mostly
enriched
within
a
central
region
of
the
protein.
Using
a
combination
of
mutagenic
analysis
and
live-­‐cell
imaging
I
demonstrate
that
Tea1
phosphorylation
might
be
required
for
its
dissociation
from
the
cluster-­‐network
at
the
cell
tip.
This
suggests
that
Tea1
interactions
within
the
cluster
network
are
phospho-­‐regulated
by
one
of
the
several
tip-­‐localized
kinases. It
has
been
shown
in
other
organisms
and
in
this
thesis
that
comparison
among
MS
samples
requires
quantitative
MS
methodologies.
Thus,
I
developed
a
robust
SILAC
(Stable
Isotope
Labeling
in
Cell
Culture)
method
to
perform
quantitative
MS
in
S.
pombe.
As
a proof-­‐of-­‐principle
of
the
method
I
performed
a
proteome-­‐wide
comparison
between
the
late
G2
and
the
G1/S
transition
of
the
cell
cycle.
The
cell
cycle
proteome-­‐wide
analysis
not
only
quantified
variation
in
expression
levels
of
cell
cycle
regulated
proteins
but
also
identified
novel
cell
cycle
regulated
proteins.It
has
been
previously
shown
that
Tea1,
Tea3
and
Mod5
can
interact
simultaneously,
with
each
pair
interacting
independently
of
the
third
protein.
I
describe
here
a
Mod5
mutagenic
analysis
screen
designed
to
separate
Tea1
and
Tea3
binding
site
on
Mod5.
The
Mod5-­‐mutants
obtained
from
this
analysis
indicate
that
the
Tea3-­‐Mod5
interaction
may
play
a
role
in
cell
polarity
establishment.
Moreover,
although
Tea3
is
non-­‐essential
for
the
cluster-­‐network
formation,
Tea3
might
be
important
for
its
compaction,
which
may
be
particularly
important
during
de
novo
formation
of
cell
polarity.